PROJECT SUMMARY Transcription inhibition has been identified as a significant weakness and exploitable target in many forms of aggressive metastatic cancer. Transcriptional kinases that regulate the early stages of transcription initiation and elongation are overexpressed in aggressive cancers such as triple-negative breast cancer and multiple myeloma to globally upregulate transcription. Preclinical and phase I clinical trials with transcriptional kinase inhibitors have shown promise for treating cancer. These kinases, CDK7 and CDK9, phosphorylate the RNA Polymerase II (Pol II) C-terminal domain (CTD) and associated factors to regulate Pol II initiation and promoter-proximal pausing (pausing). Pausing is the rate-limiting step in transcription and must be bypassed in cancer to upregulate transcription, bringing CDK7 and CDK9 to the forefront of cancer therapeutics. However, further drug develop- ment has been impeded by the lack of a defined mechanism for these CDKs. Previous experimentation failed to define the mechanistic contributions of each kinase due to 1) use of crude in vitro systems (e.g. nuclear extracts) or 2) the use of non-specific, promiscuous kinase inhibitors in cell-based assays. Here, I propose to describe the mechanism of CDK7 and CDK9 regulation of promoter-proximal pausing using a unique reconstituted in vitro transcription assay and state-of-the-art cell-based assays combined with specific, next-generation inhibitors. My project will have three distinct goals, 1) dissecting the individual contributions of CDK7 and CDK9 in pausing, 2) characterizing kinase co-regulatory events that influence pausing, and 3) evaluating the mechanism of CDK7/CDK9 pause regulation in cells. The first two will utilize the Taatjes Lab?s in vitro transcription system that is reconstituted from purified human factors. This system surpasses traditional in vitro transcription experiments in its ability to unequivocally interrogate individual factors. I will determine the independent roles of CDK7 and CDK9 in pause regulation through inhibition of each kinase in in vitro transcription assays. Coregulation and cooperation between the kinases will be evaluated with similar assays that combine specific inhibitors. Finally, I will combine precision run-on sequencing (PRO-seq) with next-generation CDK7 and CDK9 inhibitors to assess the role of these kinases in cells and validate my in vitro work. The insights gained from this experimentation will provide the mechanistic understanding needed to develop cancer therapeutics with minimal side effects.